Human basophils selectively express the FcγRII (CDw32) subtype of IgG receptor

Human basophils selectively express the FciyRII (CDw32) subtype of IgG receptor Lisa M. Anselmino, PhII,**** Bite Perussia, MD,*** and Larry L. Thomas, PhD* Chicago, 111..md Philmdelphiu, Pu. The role

(f IgG

uncerturn.

untibody

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the Fc receptorsjbr

(FcyR)

thut bind

&rali:ed

ufter

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with

hig h-c!/finifl HCwerer. profile

a rnurine Fc yRI

did

not induce

FcyRI

dcje\ not occur.

with

or u’ith MAbs with

N MAb

gtrmmcr FcyRI

is exclusive& (CD64)

‘J ALLERGY

with

suggests

and ma.st cells

by tw,o-color

incuhnted

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that rrntigenic,

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induced

OII basophils.

of‘ the FcyRII

through

the low-c#ni&

the low-@ini?

uus not nltered

iCD~,32)

FcyRIII

expre.ssion These

suhepe.

MAbs

was

Busophils

did

its Fc to the

Fc yRl1

IgE-mediated

.sensiti;ntion

antibodies

qf biotinylnted conjugate.

specificcdl~

cell

with fluorescein

monoclonal

Binding

(CD/h). (CDw32).

The

activation. of Fc yRI

results

indicate

The absence

of’ basophils

is

ana1ysi.c

mononuclear

sequentially

1gE and biotinylated

to cmtigen

fluorometric

Basophil-contnining were

FcyR

lgG2~1.

Fc yR expression

on ba.>ophrl.s

h/;:h-alfinitv

monoclontrl

monomeric

reacted

basophik.

volunteers

to the different incubation

(CD64),

pretreatment

mvtrophils, pr,v.srnt

subsequent

(f basophil

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murine

:;pecl$cally

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cmd nonatopic,

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IpG

Ct.1.~ iW.Wti.YOL 1989:84:907-14.1

The IgE-mediated sensitization of basophils and mast cells to specific antigen is clearly established as an integral event in reactions of immediate hypersensitivity.’ The occurrence of elevated serum levels of antigen-specific lgG4 antibody in association with various aller::ic diseases24 has suggested that lgG4 also may contribute to the pathogenesis of allergic disorders. Although selected subclasses of monomeric IgG antibody mediate a transient antigenic sensitization of basophils and mast cells in animals,’ the role of IgG4 antibody in the sensitization of human basophils and mast cells is uncertain. Basophils, which

Abbreviations

used

FcyR: Fc receptorfor l&G MAb: FITC: yIFN: PE: DNP: DNP,,-HSA:

Monoclonal antibody Fluorescein isothiocyanate Gamma interferon Phycoerythrin Dinitrophenyl DNP (21 mol) conjugated to human serum albumin (I mol)

PAG: PIPES-albumin buffer, pH 7.4, conNK:

taining 5.4 mmol/L of glucose Natural killer

EDTA: Ethylenediaminetetraacetlcacid Ka: Association constant From the *Department of Immunology/Microbiology. Rush Medical College, Chicago. Ill., and ***The Wistar Institute. Philadelphla, Pa. Supported by Nattonal Institutes of Health Grants A117177. CA37 155, and CA45884. Received for publication March 20, 1989. Revised July IO, 1989. Accepted for publication July 14, 1989. Reprint requests: Larry L. Thomas, PhD, Department of Immunology/Microbiology, Rush Medical College, 1653 West Congress Parkway, Chicago, IL 60612. **Submitted in partial fulfillment of the requirements for the PhD degree from the Graduate College of Rush University. Present address: Abbott Laboratories. North Chicago, IL 60064. B. Perussia. .hdD. IS a Leukemia Society Scholar. 111115392

have been studied more extensively in this context, have been demonstrated by autoradiography to possess Fc~R.~ However, efforts to demonstrate IgG-mediated histamine release from basophils have yielded conflicting results.‘-” The FcyR present on human leukocytes are classified into three functionally and antigenically distinct subtypes”: (1) a 72 kd, high-affinity receptor termed FcyRI (CD64) that is present on monocytes and inducible by ylFN, (2) a 42 kd, low-affinity receptor termed FcyRII (CDw32) that is present on a variety 907

908

Anselmino

et al.

TABLE I. Specificities

J. ALLERGY

of MAbs

Fc-gran 1 (IgGZb), and Leu-l lc (IgGl) each react with a

MAb

Class

FcyR specificity

B137.17* KuFc79 3G8 Leu-I Ic CLB Fc-gran 1

IgG2a IgG2b IgG 1 IgG 1 IgG2b

FcyRI (CD64) FcyRIl (CDw32) FcyRIII (CD 16) FcyRlII (CD16) FcyRIII (CD1 6)

*Binding to FcyRI occurs through the Fc portion of the antibody.

of leukocytes, including monocytes, neutrophils, and B-lymphocytes as well as on platelets, and (3) a 50 to 70 kd , low-affinity receptor termed FcyRIII (CD 16)

that is present on neutrophils and NK cells. The FcyRI (CD64) binds monomeric IgG with high affinity (Ka = 1-3 X 10’ mol/L-i) in a rank order of IgGl > IgG3 > IgG4 > IgG2,” and also binds murine monomeric IgG2a with high affinity. ‘I In contrast, FcyRII

(CDw32)

and FcyRIII

(CD16)

bind mono-

meric IgG much less avidly (Ka equals approximately 5 X 10’ mol/L-’ to 2 X lo6 mol/L-‘).‘3,‘4 Because basophil sensitization by monomeric IgG4 presumably

would be dependent on the presence of the highaffinity FcyRI (CD64), we postulated that delineation of the FcyR subtype(s) expressed on basophils would assist in assigning a role for IgG antibody in basophil activation. We therefore characterized the FcyR present on human basophils by two-color fluorometric analysis with a murine monomeric IgG2a that binds specifically through its Fc to the high-affinity FcyRI and MAbs specific for the two low-affinity receptors, FcyRII and FcyRIII. MATERIAL MAbs

CLIN. IMMUNOL. DECEMBER 1989

AND METHODS

The MAb B137.17 was produced by one of us (B. Perussia), and Leu-1 lc (anti-CD16) was purchased from Becton Dickinson (Mountain View, Calif.). The remaining MAbs were the generous gifts of Dr. J Unkeless (Mount Sinai Hospital, New York, N.Y.) (3G8; anti-CD16). Dr. P. Tetteroo (University of Amsterdam, The Netherlands) (CLB Fc-gran 1; antiCD16), and Drs. P. Gambel and T. Mohanakumar (Medical College of Virginia, Richmond, Va.) (KuFc79; anti-CDw32). The B137.17 is an IgG2a of unknown specificity selected on the basis of high-affinity binding in the monomeric form to the -yIFN-inducible FcyRI (CD64) on monocyte and myeloid cell lines.” The binding of B 137. I7 to FcyRI (CD64) is Fc fragment-dependent and is blocked by monomeric human IgG (order of potency: IgGI 2 IgG3 3 > IgG4 2 IgG2) and by murine IgG2a and IgG3. Sepharose-linked B 137.17 precipitates a 72 kd molecule identified as FcyRl (CD64) from human monocytes and myeloid cell lines. The KuFc79 (IgG2b) reacts with a 42 kd molecule identified as the FcyRII (CDw32) on monocytes, granulocytes, and B cells.‘” The 3G8 (IgGl),

50 to 70 kd molecule identified as FcyRIII (CD16) on neutrophils, NK cells, and macrophages.” I9 The specificities and classes of the antibodies used in the study are summarized in Table I. The MAbs B137.17, 3G8, CLB Fc-gran 1, and KuFc79 were biotinylated as described elsewhere”‘; Leu-1Ic was purchased in the PE-conjugated form. DNPspecific murine monoclonal IgE was purified from ascites of BALB/c mice after passage of hybridoma Hl DNP-a26.82 cells,” which had generously been provided by Drs. F-T. Liu and D. Katz (Medical Biology Institute, La Jolla, Calif.). The IgE anti-DNP was conjugated with FITC by Dr. W. Gathings (Southern Biotechnology Associates, Homewood, Ala.).

Mononuclear

cell preparation

The basophil-containing mononuclear fraction was isolated from venous blood of healthy adult volunteers known to have >l% circulating basophils. The blood was diluted 1:4 in 0.15 mol/L of NaCl containing 4 mmol/L of EDTA, and 30 ml aliquots were overlayed onto 10 ml cushions of Ficoll-Hypaque (density, 1.080). The tubes were centrifuged at 300 g for 30 minutes (22” C). The mononuclear cell fraction was harvested and washed two times in icecold buffer (PAG) containing 25 mmol/L of piperazineN,N’-bis-(2-ethane sulfonic acid) (Sigma Chemical Co.. St. Louis, MO.), 110 mmol/L NaCI, 5 mmol/L KCI, 40 mmol/L NaOH, 0.003% HSA, and 5.4 mmol/L glucose (pH 7.4). The cells were collected by centrifugation at 200 g for 10 minutes at 4” C. The cells then were suspended in IO ml ice-cold 0.01 mol/L of lactic acid, 0.15 mol/L of NaCl (pH 3.9), and held at 4” C for 5 minuts to elute native IgE.22 After addition of 30 ml of cold PAG, the cells were pelleted by centrifugation at 200 g for 10 minutes (4” C) and were washed two times in cold PAG. The percentage of basophils in the cell preparation was measured by alcian blue staining. For some experiments as indicated, blood was diluted 1 :4 in 0.075 mol/L of NaCl before the FicollHypaque density centrifugation. This modification enriched the basophil percentage in the cell preparations of some donors (Thomas L. Unpublished observation).

Incubation with MAbs and two-color fluorometric analysis The lactic acid-treated cells were suspended in 0.5 ml of 50 Pg/ml of FITC-IgE anti-DNP in PAG buffer containing 4 mmol/L of EDTA and 10 pg/ml of heparin and were incubated for 20 minutes at 37” C. The cell suspension was diluted by addition of 20 ml of cold PAG, and the cells were collected by centrifugation at 200 g for 10 minutes (4” C). The cells were washed with cold PAG, and aliquots containing IO6 cells were added to 1.5 ml conical microcentrifuge tubes for incubation with the MAbs. The cells were pelleted by centrifugation at 200 g for IO minutes (4” C), and 0.02 ml of biotin-labeled B137.17 (25 pg/ml), 3G8 (20 pg/ml), CLB Fc-gran 1 (20 pg/ml), or KuFc79 (20 kg/ml) or 0.02 ml PE-labeled Leu-llc (50 kg/ml) was added. Preliminary experiments with positive control cells had established that the MAbs and the monomeric IgG2a

VOLUME NUMBER

Basopnil Fc$II (CDw32) expression

84 6. PAPT !

were used at saturating concentrations. The mixtures were incubated for 30 minutes on ice and were washed once with phosphate-buffered saline (pH 7.4) containing 0. I’% gelatin and 0. I % sodium azide. With the exception of cells that had been incubated with Leu-I lc, the cells were incubated subsequently with 0.02 ml of PE-strepavidin conjugate (SO p,g/ml) for 15 minutes on ice. The cells were washed once with cold PAG containing 4 mmol/L of EDTA and were resuspended in 0.2 ml of the same buffer for two-color Ruorometric analysis. Integral log green (FITC) fluorescence and integral log red (PE,) Ruorescence were measured with an Epics C flow cytometer (Coulter Electronics, Hialeah. Fla.) equipped with ,I 5 W argon laser (488 nm excitation. 300 mW emission). For each sample, 5000 cells were analyzed. and fluorescence intensity was displayed on a 256 channel. 3-decade log scale delineated in arbitrary log units. Overlap of PE and FITC spectral emissions was climinated by clectronrc compensation. Background PE Huorescencc was established with cells incubated with FITCIgE anti-DNI’ and PE-strepavidin conjugate. Basophil percentages determined by Ilow cytometric analysis corrcsponded to tht: basophil percentages determined by alcian blue staminp.

Histamine

release

Basophils passively sensitized as described above with FITC-IgE anti-DNP were incubated with 4 ngiml of DNP,,HSA” in PAG buffer containing 0.6 mmol/L of CaCIZ and I mmoi 1L of MgC12 for 30 minutes at 37” C. Incubation volume was 0 5 ml and contained 5 x 10’ cells. Reactions were stopped by centrifugation for 2 minutes at 1000 K, and histamine release was measured as described previously.” Spontaneous histamine release was determined with cells incubated in the absence of DNP,,-HSA and was <5%,

Treatment

with

$FN

Basophil-containing mononuclear cells were isolated and incubated with FITC-IgE anti-DNP under sterile conditions as described above. Neutrophils were recovered sterilely from the post-Ficoll-Hypaque centrifugation cell pellet as described clsc:whcre.‘r After the cells were washed once in RPMI 1640. the mononuclear cells and neutrophils were each suspended at 4 x 10hcells per milliliter in RPM1 1640 containing 10% fetal calf serum alone or with 200 U/ml of human recombinant yIFN (Dr. H. Shephard, Genentech. Inc.. South San Franciso, Calif.). One milliliter aliquots were added to 24.well tissue-culture plates (Costar Corp.. Cambridge. hilass. ), and the cell suspensions were incubated for 18 hours at 37” C in 5% CO?. The cells were collected by centrifugation at 200 g for IO minutes (4” C) and were resuspended at IO” cells per milliliter in PAC. Incubation with the Fc?R-specific MAbs and the monomeric IgG2a was performed a\ described above.

RESULTS Identification

of basophil

FcyR

Previous studies in our laboratory had established that incubation of mononuclear cell preparations with

909

FITC-IgE anti-DNP resulted in selecti\r hindmg of the FITC-IgE to the basophils.” Therefore. to identify the basophil FcyR subtype(s). aliquots c:f basophilcontaining mononuclear cells that had first been incubated with FITC-IgE anti-DNP were Incubated sequentially with the biotin-labeled FcyR-specific MAbs or monomeric IgG2a and with PE-strepavidin ,-HSA in PAG containing 0.6 mm& I. of CaCI, and 1 mmolil~ of VgC’l, for 4

910

Anselmino

et al.

J. ALLERGY

8137.17

CLIN. IMMUNOL. DECEMBER 1989

KuFc79

CLB Fc-granl

LOGGREENFLUURESCENCE FIG. 1. Two-color histogram of basophil reactivity with FcyR-specific MAbs. Results of one experiment with cells of a single donor enriched to 44% basophils are presented. Binding of FITC-IgE anti-DNP is indicated by log green fluorescence intensity, and binding of IgGPa or the FcyR-specific MAbs is indicated by log red (PE) fluorescence intensity; quadrant 1, PE-positive and FITC-negative cells; quadrant 2, PE-positive and FITC-positive cells; quadrant 3 (not labeled), PE-negative and FITC-negative cells; quadrant 4, PE-negative and FITC-positive cells.

minutes at 37” C. The reactions were stopped by addition of cold PAG, the cells were washed once with cold PAG, and the cells then were incubated as above with the IgG2a B137.17 or with 3G8 (anti-CD16). The results presented in Fig. 2 demonstrate that cells stimulated with the DNP,,-HSA displayed the same reactivity as control ceils; control cells also had been sensitized with FITC-IgE but were not stimulated with DNP,,-HSA. In each case, the cells that bound either the IgG2a B137.17 or the anti-CD16 3G8 were restricted to quadrant 1 and did not bind FITC-IgE antiDNP. In the same experiment, basophil reactivity with the anti-CDw32 KuFc79 (quadrant 2) also was unchanged after stimulation with the DNP,,-HSA. Similar results were obtained in three additional experiments with cells of different donors, including one

atopic individual. Preincubation with DNP,,-HSA also did not alter the staining patterns with CLB Fcgran 1 (anti-CD16) or Leu-llc (anti-CD16) (results not presented). Incubating aliquots of the cells with 4 rig/ml DNP,,-HSA for 30 minutes at 37” C stimulated 6% to 37% net histamine release in the experiments . Effect of ylFN treatment FcyRl expression

on

Preincubation with yIFN induces expression of the high-affinity FcyRI (CD64) on human neutrophils.26 Therefore, experiments were performed to determine if exposure to yIFN could induce FcyRI (CD64) expression on basophils. Results presented in Fig. 3 demonstrated that incubation of mononuclear cell

VOLUME NUMBER

Basophil

84 6, PART 1

FcyRll (CDw32) expression

911

CONTROL KuFc79

368

B137,17

+DNP2yHSA KuFc79

368

B137.17

2

1 : I..” .r. I 75. : : if :. 5. :.:,: .. %:a: . gAc”:.’ ~. ‘“Y . . .. : :.::.I;,

4

LOGGREENFLUORESCENCE FlfG. 2. Influence of IgE-mediated activation on basophil expression of Fc-yRl (CD64). Fc$W (CDw32), and Fc-yRlll (CD16). Cells (5% basophils) sensitized with FITC-IgE anti-DNP were incubated in buffer with (lower panels) or without (upper panels) 4 ng /ml DNP,,-HSA for 4 minutes at 37” C before incubation with the lgG2a 8137.17, anti-CD16 3G8, or anti-CDw32 KuFc79. Fluorescence and quadrants denote the same parameters as described for Fig. 1,

fractions with 200 U I ml of human recombinant yIFN for 18 hours at 37” C did not result in basophil binding of the IgG2a B 137.17. In the same experiment, 54% of the neutrophils isolated from the same donor bound B137. I7 after incubation with yIFN under the same conditions (results not presented). In the absence of YIFN, 0.5% of the neutrophil population bound the IgG2a B 137.17. Similar results were obtained in two additional experiments, each with cells of a different donor. Exposure of neutrophils to the lactic acid solution used to elute native IgE off the basophils did not impair induction of FcyRI (CD64) on neutrophils by yIFN (results not presented). DISCUSSION These results demonstrate that the FcyR on human basophil@ IS exclusively of the low-affinity FcyRII (CDw32) subtype. Basophils did not react with the IgG2a B 137.17, which binds specifically through its Fc to the 7:! kd high-affinity FcyRI (CD64),” or with 3G8, CLB Fc-gran 1, or Leu-1 lc, each of which is

specific for the 50 to 70 kd low-affinity FcyRIII (CD16).“-19 In contrast, KuFc79, which is specific for the 42 kd low-affinity FcyRII (CDw32) common to various leukocytes,“’ I6 reacted with the entire basophi1 population of each donor tested. Based on the relative intensity of the PE fluorescence after incubation with KuFc79, the density of FcyRII (CDw32) on basophils is similar to the density on monocytes (Figs. 1 and 2) and on neutrophils (Thomas L, Perussia B. Unpublished observation). As such, these results corroborate and provide a basis for the earlier autoradiographic data that demonstrated that aggregated, but not monomeric, IgG bound to the FcyR on basophils.6 The failure of the IgG2a B 137.17, which binds specifically to the FcyRI (CD64), and of the MAbs specific for the FcyRIII (CDl6) to react with basophils could not be attributed to an adverse effect of the lactic acid treatment or to a steric hindrance by the FITC-IgE. The IgG2a and MAbs exhibited appropriate reactivities with other cells present in the lactic acid-treated cell preparations and reversal of the

912

Anselmino

et al.

J. ALLERGY

CONTROL

CLIN. IMMUNOL. DECEMBER 1989

+ rIFN

LOGGREEN FLUORESCENCE FIG. 3. Influence of ylFN on basophil expression of high-affinity FcyRl (CD64). Cells (6% basophils) that had been preincubated with FITC-IgE anti-DNP were incubated for 16 hours at 37” C in the presence or absence of 200 U/ml of human recombinant ylFN before incubation with the monomeric lgG2a 8137.17. The Y axis indicates log red (PE) fluorescence for 8137.17 binding. Quadrants are as described for Fig. 1.

order of incubation (i.e., FcyR-specific MAbs or monomeric IgG2a before FITC-IgE) did not alter the reactivity profile of basophils. The hypotonic conditions usedto enrich for basophilsin someexperiments also did not influence expression of the FcyR, since the same reactivity profiles were obtained with basophils that had not been exposed to the hypotonic conditions. Another laboratory also has reported recently that basophils express FcyRII (CDw32) but lack FcyRIII (CD16).*’ In that study, basophils were identified cytochemically, and a different FcyRIIspecific (anti-CDw32) MAb was used. In addition to establishing that basophils also lack FcyRI (CD64), the present results indicate that the profile for FcyR expressionis similar for basophils of atopic and nonatopic individuals. Furthermore, the results demonstrate that brief (4 minutes) IgEmediated activation of the basophils does not lead to expression of FcyRI (CD64) or FcyRIII (CD16) and does not increase expression of FcyRII (CDw32). Therefore, these findings indicate that neither the immediatemembraneand cytoskeletal changes**nor the early biochemical events’,29initiated by IgE crossbridging alters FcyR expression on basophils. The possibility that a longer duration of IgE-mediated activation might alter FcyR presentationcannot be excluded. Short-term (8 to 16 hours) exposure to yIFN increasesexpression of FcyRI (CD64) on monocytes and macrophages3’and inducesFcyRI (CD64) expression on neutrophils, which normally are devoid of Fc~RI.*~However, as presentedhere, yIFN at a con-

centration that was optimal for induction of FcyRI (CD64) on neutrophils did not induce FcyRI (CD64) expression on basophils. A study that yIFN induces expression of HLA-DR on basophils2’ suggeststhat lack of basophil responsivenessto yIFN is not the basis for the absence of FcyRI (CD64) induction. However, the results do not exclude the possibility that other mediatorsmay induce expressionof FcyRI, or additionally FcyRIII, on basophils. The increased levels of IgG4 antibody associated with various allergic disorders2Aand the homocytotropic property of selectedsubclassesof IgG antibody in animal9 has suggestedthat IgG4 may sensitize human basophils and mast cells to specific antigen. Consistent with this postulate, a human IgG fraction was demonstratedto produce short-term sensitization of monkey skin,3’ and polyclonal anti-IgG was demonstrated to stimulate human basophil histamine release.’ Subsequentresults obtained with MAbs directed againstthe individual subclassesof human IgG demonstratedthat basophil histamine releasewas initiated only during incubation with anti-IgG49;the histamine releasewas measuredin the presenceof deuterium oxide, which hasbeendemonstratedto enhance IgE-mediated histamine release.32In another study, a MAb againsthuman IgG4 also stimulated a low level of histaminereleasein the absenceof deuterium oxide from basophils of one of three donors tested.” However, incubation with human myeloma IgG49or IgG4rich serum” failed to sensitizebasophils to anti-IgG4 or to specific antigen. Results of other laboratories also have indicated against IgG4-mediated histamine

VOLUME NUMBER

Basophil

84 6, PART 1

release from human basophils’ as well as from cutaneous mast cells.d The lack of l;he high-affinity FcyRI (CD64) suggests that it is unlikely that monomeric IgG4, or any IgG subclass in monomeric form, sensitizes basophils to specific antigen. Instead, the selective expression of FcyRlI (CDw32), which has a much lower affinity for IgG (Ka equals approximately 2 X lOh mol/L ‘)” than does FcyRI (CD64) (Ka equals approximately lo* mol/L,-‘),I’ suggests that only complexed or aggregated IgG is capable of binding to basophils. Binding measurements performed with the individua! IgG subclasses and COS cells expressing recombinant FcyRII (CDw32) yielded a rank order of 1gG1 > IgG2 = IgG4 > > IgG3. ” These findings raise the possibility that, with the possible exception of lgG3, oligomeric IgG formed by complexing with antigen may sensitize basophils to subsequent antigen exposure if any antigen binding sites on the IgG remain available. Indeed, results obtained with KuFc7’3 (anti-CDw32) stimulation of monocytes” and neutrophils’4 have established that FcyRII (CDw32) can mediate cellular activation. However, heat-aggregated IgG did not stimulate basophil histamine release,” which suggests alternatively that oligomeric IgG may act to influence other basophilactivation mechanisms. i,n either case, the findings presented here indicate that any role for IgG in allergic disorders, at least with regard to basophil-associated events. is mediated by FcyRII (CDw32) and thus limited to an effect of oligomeric or complexed IgG. Note added in proof. Another laboratory also has reported recently the presence of FcyRII (CD64) on basophils?

7

x

9

IO

11 12

13

14 15

16 17

18

We thank Michelle Haskell and Susan Arra for technical assistance and Mary Rolfe-Shaw for help in preparation of the manuscript.

19

REFERENCE!3

20

I Ishizaka 1. Mechanisms of IgE-mediated hypersensitivity. In:

2 3. 4.

5. 6.

Middleton E Jr. Reed CE, Ellis EF, Adkinson NF Jr, Yuninger JW, eds. Allergy: principles and practice. 3rd ed. St. Louis: CV Mosby, 1988:7 1. Aalberse RC, Van der Gaag R, Van Leeuwen J. Serologic aspects of IgG4 antibodies. J Immunol 1983;130:722. Merret J, Burr ML, Merret TG. A community survey of IgG4 antibody evels. Clin Allergy 1983;13:397. Rowntree S, Platts-Mills TAE, Cogswell JJ, Mitchell EB. A subclass IgG4-specific antigen-binding radiommunoassay (RIA): ccmparison between IgG and IgG4 antibodies to food and inhaled antigens in adult atopic dermatitis after deaensitization treatment and during development of antibody responses in children. J ALLERGYCLIN IMMUNOL1987;80:622. Spiegelberg HL. Biological activities of immunglobulins of different classes and subclasses. Adv Immunol 1974;19:259. Ishizaka T, Sterk AR, Iahizaka K. Demonstration of Fc re-

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FcrRll (CDw32) expression

913

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Anselmino

27.

28.

29.

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Glandular secretion of lactoferrin in a patient with neutrophil lactoferrin deficiency Gordon D. Raphael, MD,* Janet L. Davis, MD,** Philip C. Fox, DDS,*** Harry L. Malech, MD,* John I. Gallin, MD,* James N. Baraniuk, MD,* and Michael A. Kaliner, MD* Bethesda, Md. Patients with spec$c granule dejiciency (SGD) develop recurrent severe bacterial skin infections. Neutrophils from patients with SGD are dejicient in lactoferrin (Lf), an antimicrobial protein commonly found in many mucosal secretions. Unstimulated and stimulated nasal secretions, saliva, and tears were collected from a patient with SGD and from normal control subjects and were analyzed for Lf. The secretions from the patient contained normal values of Lf, suggesting that the glands secrete Lf from a source other than neutrophils. Immunohistochemical staining of normal nasal mucosa demonstrated that Lf is localized within serous submucosal gland cells and that neutrophils are not normally observed in the nasal mucosa. These findings suggest that glandular tissues produce and locally secrete Lf by processes that are independent of neutrophil degranulation. (J ALLERGY CLIN IMMUNOL 1989;84:914-9.)

From the *Allergic DiseasesSection and Bacterial DiseasesSection, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, **Laboratory of Immunology, National Eye Institute, and ***Clinical Studies Unit, Clinical Investigations and Patient Care Branch, National Institute of Dental Research,Bethesda,Md. Received for publication March 20, 1989. Revised July 11, 1989. Accepted for publication July 14, 1989. Reprint requests:Gordon Raphael, MD, The National Institutes of Health, 9000 Rockville Pike, Bldg. 10, 1lC207, Bethesda,MD 20892. l/1/15393

914

Abbreviations used Lf: Lactoferrin SGD: Specific granule deficiency PT: Phosphate-buffered saline with Tween

The submucosal glands protect the upper respiratory tract against infection by a variety of methods. Mucus production helps remove inhaled foreign particles and lubricates the mucosal surface. The nasal